The understanding of the etiology of muscular dystrophy currently lacks an in-depth, molecular description. This proposal focuses on the structural and functional studies of dysferlin, its interaction with phospholipid membranes, and the effects of disease-causing mutations on its function. Dysferlin is a 237 kDa, multi-C2 domain, integral membrane protein that is involved in membrane repair in skeletal muscle tissue. Mutations in dysferlin have been linked to Limb-Girdle Muscular Dystrophy (LGMD) in humans. Therefore, our main objective in this application is to assemble a complete 3D structure of dysferlin using electron microscopy (EM), X-ray crystallographic and biochemical techniques to develop a molecular description for how mutations impair dysferlin' s function as a mediator in membrane repair. The central hypothesis of this proposal is based on the idea that each of the seven C2 domains in dysferlin serve a unique role, and that normal dysferlin function depends on the correct spatial arrangement of its C2 domains. Guided by strong preliminary data, this hypothesis will be tested by pursuing three Specific Aims: (i) to understand the domain organization of the full- length dysferlin protein, and its relationship with MG53, we will assemble a medium-resolution structure of the dysferlin protein and MG53 using EM techniques. We have preliminary images of both dysferlin and MG53, and we have begun the process to assemble individual orientations into a single 3D structure. In addition, we will measure the function of dysferlin using a novel EM-based lipid aggregation assay; (ii) to study the individual domains of dysferlin at higher resolution, we will pursue the crystal structures of the isolated C2 and FerA domains. We have recently solved the crystal structures of the C2A domain of dysferlin to 2.0 angstroms and a novel splice variant of C2A to 1.7 angstrom resolutions. The remaining C2 domains have been isolated and purified. The central FerA domain is a four-helix bundle, and may be related to the membrane fusion activity of dysferlin. (iii) to understand the effects of disease-causing mutations on the structure and function of dysferlin, we have mapped 40 clinically isolated point mutations to the C2 domains of dysferlin. These mutations clustered into three structural categories: the Ca2+/phospholipid binding pocket, the structural -sheets of the molecule, and the effector binding region of the C2 domains. Representative mutations from each category, and three truncation mutations will be studied by EM for their effect on the total structure and function of dysferlin. In addition, representative mutations will be studied using spectroscopic techniques to understand the effects of mutation on ligand binding and domain stability. This approach is innovative, as it brings to bear state-of-the-art EM techniques with X-ray crystallography and a background in C2 domain structural biology to better understand the biology of a debilitating human disease. Once completed, a 3D structure of dysferlin could ultimately be used for domain-targeted drugs, as well as in the design of smaller, functional dysferlin proteins that are more amenable to current gene therapy techniques for the treatment of LGMD.